We aim to explain the neural origins of breathing behavior. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project focuses on the core rhythm-generating circuit for inspiratory breathing movements in the pre-Btzinger complex (preBtC) of the brainstem. The preBtC has been studied for 25 years. Its defining characteristics and its constituent rhythm-generating neurons have been documented in some detail, however, the molecular identity of the ion channels in preBtC neurons that generate inspiratory bursts remains unidentified. The specific aims of this proposal address this key knowledge gap. If we succeed then breathing would be the first mammalian motor behavior that can be explained at the level of networks, cells, and ion channels. In particular, this projec will test one leading hypothesis that a class of non-selective cation channels generates inspiratory bursts, and by extension, that these channels underlie the motor drive for breathing in living animals. It is widely acknowledged that a molecularly unidentified Ca2+-activated non-selective cationic current (ICAN) contributes to inspiratory burst generation in preBtC neurons. Non-selective cation channels of the transient receptor potential (TRP) superfamily are the best candidates for the channel(s) that give rise to ICAN in the preBtC based on evidence from several sources. We have three main objectives. 1) Determine which TRP channels are expressed in preBtC neurons using quantitative PCR, physiology, and immunohistochemistry. 2) Down-regulate these TRP channels by interfering with mRNA transcripts and overexpressing dominant negative channel mutants in rhythmically active slice cultures. Then, we will measure the degree to which the burst-generating capability of preBtC neurons is compromised. 3) Down-regulate the same TRP channels in the preBtC of living mice and measure the extent to which breathing movements are impaired. This project innovates by using contemporary molecular biology techniques to address a problem that has heretofore been approached primarily by electrophysiology. This project is significant because it identifies the ion channels responsible for inspiratory burst generation, aggregate motor drive, and real breathing behavior, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who firt characterized ICAN as well as the genetic identity of the core rhythmogenic preBtC neurons. If this project succeeds, then we will know the molecular-level mechanism for inspiratory burst generation. Medical science would then be able to therapeutically target those channels to ameliorate respiratory pathologies in many circumstances. Neuroscience would finally know the fundamental molecular point of origin for all respiratory physiology.